Machine Learning for Clinical Decision Support of Acute Streptococcal Pharyngitis: A Pilot Study.

BACKGROUND: Group A Streptococcus (GAS) is the predominant bacterial pathogen of pharyngitis in children. However, distinguishing GAS from viral pharyngitis is sometimes difficult. Unnecessary antibiotic use contributes to unwanted side effects, such as allergic reactions and diarrhea. It also may increase antibiotic resistance. OBJECTIVES: To evaluate the effect of a machine learning algorithm on the clinical evaluation of bacterial pharyngitis in children. METHODS: We assessed 54 children aged 2-17 years who presented to a primary healthcare clinic with a sore throat and fever over 38°C from 1 November 2021 to 30 April 2022. All children were tested with a streptococcal rapid antigen detection test (RADT). If negative, a throat culture was performed. Children with a positive RADT or throat culture were considered GAS-positive and treated antibiotically for 10 days, as per guidelines. Children with negative RADT tests throat cultures were considered positive for viral pharyngitis. The children were allocated into two groups: Group A streptococcal pharyngitis (GAS-P) (n=36) and viral pharyngitis (n=18). All patients underwent a McIsaac score evaluation. A linear support vector machine algorithm was used for classification. RESULTS: The machine learning algorithm resulted in a positive predictive value of 80.6 % (27 of 36) for GAS-P infection. The false discovery rates for GAS-P infection were 19.4 % (7 of 36). CONCLUSIONS: Applying the machine-learning strategy resulted in a high positive predictive value for the detection of streptococcal pharyngitis and can contribute as a medical decision aid in the diagnosis and treatment of GAS-P.

Rapid expansion and international spread of M1UK in the post-pandemic UK upsurge of Streptococcus pyogenes.

The UK observed a marked increase in scarlet fever and invasive group A streptococcal infection in 2022 with severe outcomes in children and similar trends worldwide. Here we report lineage M1UK to be the dominant source of invasive infections in this upsurge. Compared with ancestral M1global strains, invasive M1UK strains exhibit reduced genomic diversity and fewer mutations in two-component regulator genes covRS. The emergence of M1UK is dated to 2008. Following a bottleneck coinciding with the COVID-19 pandemic, three emergent M1UK clades underwent rapid nationwide expansion, despite lack of detection in previous years. All M1UK isolates thus-far sequenced globally have a phylogenetic origin in the UK, with dispersal of the new clades in Europe. While waning immunity may promote streptococcal epidemics, the genetic features of M1UK point to a fitness advantage in pathogenicity, and a striking ability to persist through population bottlenecks.

Toxic Shock Syndrome (TSS) Caused by Group A Streptococcus: Novel Insights Within the Context of a Familiar Clinical Syndrome.

The emergence of invasive infections attributed to group A Streptococcus (GAS) infections, has resurged since the 1980s. The recent surge in reports of toxic shock syndrome due to GAS in Japan in 2024, while sensationalized in the media, does not represent a novel infectious disease per se, as its diagnosis, treatment, and prevention are already well-established. However, due to signs of increasing incidence since 2011, further research is needed. Health authorities in neighboring countries like The Republic of Korea should not only issue travel advisories but also establish meticulous surveillance systems and initiate epidemiological studies on the genotypic variations of this disease while awaiting various epidemiological research findings from Japan.

Eliminate all risks: A call to reexamine the link between canine scabies and rheumatic heart disease.

Rheumatic heart disease (RHD) and acute rheumatic fever (ARF) disproportionately affect individuals in low-resource settings. ARF is attributed to an immune response to Group A Streptococcus (GAS) following GAS pharyngitis and potentially GAS impetigo in which infection can be initiated by scabies infestation. The burden of ARF and RHD in Aboriginal and Torres Strait Islander people in Australia is among the highest globally. Following recent calls to include dog management programs in ARF and RHD prevention programs, we believe it is timely to assess the evidence for this, particularly since previous recommendations excluded resources to prevent zoonotic canine scabies. While phylogenetic analyses have suggested that the Sarcoptes mite is host specific, they have differed in interpretation of the strength of their findings regarding species cross-over and the need for canine scabies control to prevent human itch. Given that there is also indication from case reports that canine scabies leads to human itch, we propose that further investigation of the potential burden of zoonotic canine scabies and intervention trials of canine scabies prevention on the incidence of impetigo are warranted. Considering the devastating impacts of ARF and RHD, evidence is required to support policy to eliminate all risk factors.

Using 16s rRNA sequencing to characterize the microbiome of tropical cutaneous ulcer disease: insights into the microbial landscape and implications for diagnosis and treatment.

Cutaneous ulcers are common in yaws-endemic areas. Although often attributed to 'Treponema pallidum subsp. pertenue' and Haemophilus ducreyi, quantitative PCR has highlighted a significant proportion of these ulcers are negative for both pathogens and are considered idiopathic. This is a retrospective analysis utilising existing 16S rRNA sequencing data from two independent yaws studies that took place in Ghana and the Solomon Islands. We characterized bacterial diversity in 38 samples to identify potential causative agents for idiopathic cutaneous ulcers. We identified a diverse bacterial profile, including Arcanobacterium haemolyticum, Campylobacter concisus, Corynebacterium diphtheriae, Staphylococcus spp. and Streptococcus pyogenes, consistent with findings from previous cutaneous ulcer microbiome studies. No single bacterial species was universally present across all samples. The most prevalent bacterium, Campylobacter ureolyticus, appeared in 42% of samples, suggesting a multifactorial aetiology for cutaneous ulcers in yaws-endemic areas. This study emphasizes the need for a nuanced understanding of potential causative agents. The findings prompt further exploration into the intricate microbial interactions contributing to idiopathic yaw-like ulcers, guiding future research toward comprehensive diagnostic and therapeutic strategies.

Increase in invasive group A streptococcal infections (iGAS) in children and older adults, Norway, 2022 to 2024.

At the end of 2022 and most notably during the first half of 2023, the number of invasive group A streptococcus (iGAS) notifications increased in Norway, largely affecting children younger than 10 years, as observed in several other countries. Following this atypical season, a new surge in the number of iGAS notifications began in December 2023 and peaked between January and February 2024, now particularly affecting both children younger than 10 years and older adults (70 years and above).

Disruption of IL-17-mediated immunosurveillance in the respiratory mucosa results in invasive Streptococcus pyogenes infection.

Introduction: Streptococcus pyogenes is a Gram-positive pathogen that causes a significant global burden of skin pyoderma and pharyngitis. In some cases, infection can lead to severe invasive streptococcal diseases. Previous studies have shown that IL-17 deficiency in mice (IL-17-/-) can reduce S. pyogenes clearance from the mucosal surfaces. However, the effect of IL-17 on the development of severe invasive streptococcal disease has not yet been assessed. Methods: Here, we modeled single or repeated non-lethal intranasal (IN) S. pyogenes M1 strain infections in immunocompetent and IL-17-/- mice to assess bacterial colonization following a final IN or skin challenge. Results: Immunocompetent mice that received a single S. pyogenes infection showed long-lasting immunity to subsequent IN infection, and no bacteria were detected in the lymph nodes or spleens. However, in the absence of IL-17, a single IN infection resulted in dissemination of S. pyogenes to the lymphoid organs, which was accentuated by repeated IN infections. In contrast to what was observed in the respiratory mucosa, skin immunity did not correlate with the systemic levels of IL-17. Instead, it was found to be associated with the activation of germinal center responses and accumulation of neutrophils in the spleen. Discussion: Our results demonstrated that IL-17 plays a critical role in preventing invasive disease following S. pyogenes infection of the respiratory tract.

Group A Streptococcus Carriage in Adults in Southern Israel: A Prospective Study.

BACKGROUND: The prevalence of Group A streptococcus (GAS) carriage among adults is studied less than in children. The variability of reported carriage rates is considerably large and differs among diverse geographic areas and populations. OBJECTIVES: To evaluate the prevalence of GAS carriage among adults in Israel. METHODS: In this prospective study, conducted in a large healthcare maintenance organization in Israel, we obtained pharyngeal cultures from adults attending the clinic without upper respiratory tract complaints or fever. Patient data included sex, age, number of children, and religious sectors. RESULTS: From May to December 2022, eight family physicians collected a total of 172 throat swabs (86% response rate). The median age was 37 years (range 18-65); 72.7% were females, 22.7% were ultra-Orthodox Jewish, and 69.2% had children. The prevalence of GAS carriage was 6.98%, 95% confidence interval (95%CI) 3.7%-11.9%. GAS carriers were younger (31.7 vs. 39.3 years, P = 0.046), and the majority were ultra-Orthodox Jews (58.3% vs. 20%, P = 0.006). All GAS carriers were from lower socioeconomic status. When assessing risk factors for GAS carriage using multivariate analysis, only being an ultra-Orthodox Jew was positively related to GAS carriage (adjusted odds ratio 5.6, 95%CI 1.67-18.8). CONCLUSIONS: Being an ultra-Orthodox Jew was the single variable associated with a GAS carriage, which may be related to having many children at home and living in overcrowded areas. Primary care physicians in Israel should recognize this situation when examining patients with sore throats, mainly ultra-Orthodox Jews.

Detection of sgRNA via SHERLOCK as Potential CRISPR Related Gene Doping Control Strategy.

Apprehensions about gene doping have grown consistently due to advancements in gene engineering techniques, particularly with the emergence of clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based tools. These tools not only provide unprecedented possibilities for illicit performance enhancement by athletes but also offer new avenues for the detection of gene doping through biosensing of nucleic acids. Hence, pursuing on a previous study, an analytical method based on reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection by means of Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) was optimized for the direct detection of sgRNA associated with Streptococcus pyogenes in serum. Detection device, assay parameters, and sample handling were adjusted, to overcome previously determined assay limitations. The conducted method characterization confirmed the methods' specificity and increased detection sensitivity from 100 pM to 1 fM sgRNA in 100 µL of serum. Furthermore, reanalysis of in vivo mouse administration samples collected in a previous proof-of-concept study was conducted with successful identification of sgRNA in all anticipated postadministration samples within the 24-h collection period. Those findings support the applicability of the refined analytical procedure for the detection of illegal doping attempts via ribonucleoprotein-based CRISPR/Cas application through sgRNA identification, offering a new potential doping control strategy for CRISPR related gene doping.

Outbreak of Streptococcus pyogenes emm89 ST646 in a head and neck surgical oncology ward.

Streptococcus pyogenes causes a variety of human infections, and hospital outbreaks with this pathogen have also been reported. The purpose of this study is to describe the clinical characteristics of an outbreak of S. pyogenes involving 15 patients and four healthcare workers (HCWs), as well as the molecular characteristics of the causative isolates. The course and response to the outbreak were reviewed, and information on the characteristics of the patients was extracted retrospectively from the medical records. Whole-genome sequencing of the 16 causative isolates (14 from patients and two from HCWs) was also performed. All 15 patients were postoperative of head and neck cancer with tracheotomy, and 12 had invasive infections, primarily surgical site infections, all of which resolved without causing serious illness. All but the first case was detected more than 7 days after admission. S. pyogenes was detected in two patients after empiric antimicrobial administration was performed on all inpatients and HCWs, and the outbreak was finally contained in approximately 2 months. All isolates detected in patients and HCWs belonged to emm89/clade 3, a hypervirulent clone that has emerged worldwide and was classified as sequence type 646. These isolates had single nucleotide polymorphism (SNP) differences of zero to one, indicating clonal transmission. This study demonstrated an outbreak of S. pyogenes emm89/clade 3 in a ward of patients with head and neck cancer. The global emergence of hypervirulent isolates may increase the risk of outbreaks among high-risk patients. IMPORTANCE: This study describes an outbreak of Streptococcus pyogenes that occurred in a ward caring for patients with head and neck cancer and tracheostomies. Many cases of invasive infections occurred in a short period, and extensive empiric antimicrobial administration on patients and healthcare workers was performed to control the outbreak. Whole-genome sequencing analysis of the causative strains confirmed that it was a monoclonal transmission of strains belonging to emm89/clade 3. The epidemiology and clinical characteristics of S. pyogenes infections have changed with the replacement of the prevalent clones worldwide. In the 1980s, there was a reemergence of S. pyogenes infections in high-income countries due to the spread of hypervirulent emm1 strains. emm89/clade 3 has recently been spreading worldwide and shares common features with emm1, including increased production of two toxins, NADase, and streptolysin O. The outbreak reported here may reflect the high spreading potential and virulence of emm89/clade 3.

Recombinational exchange of M-fibril and T-pilus genes generates extensive cell surface diversity in the global group A Streptococcus population.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

Overlapping Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis household transmission and mobile genetic element exchange.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) and Streptococcus pyogenes share skin and throat niches with extensive genomic homology and horizontal gene transfer (HGT) possibly underlying shared disease phenotypes. It is unknown if cross-species transmission interaction occurs. Here, we conduct a genomic analysis of a longitudinal household survey in remote Australian First Nations communities for patterns of cross-species transmission interaction and HGT. Collected from 4547 person-consultations, we analyse 294 SDSE and 315 S. pyogenes genomes. We find SDSE and S. pyogenes transmission intersects extensively among households and show that patterns of co-occurrence and transmission links are consistent with independent transmission without inter-species interference. We identify at least one of three near-identical cross-species mobile genetic elements (MGEs) carrying antimicrobial resistance or streptodornase virulence genes in 55 (19%) SDSE and 23 (7%) S. pyogenes isolates. These findings demonstrate co-circulation of both pathogens and HGT in communities with a high burden of streptococcal disease, supporting a need to integrate SDSE and S. pyogenes surveillance and control efforts.

Outbreak of invasive Group A streptococcus disease in a nursing home in Ireland in February 2023 caused by emm type 18.

An out-of-season increase in cases of invasive Group A streptococcus (iGAS) was observed in Ireland between October 2022 and August 2023. We describe the management of an iGAS outbreak involving three nursing home residents in Ireland in early 2023. A regional Department of Public Health was notified of an iGAS case in a nursing home resident in January 2023. When two further cases among residents were notified 7 days later, an outbreak was declared. Surveillance for GAS/iGAS infection in residents and staff was undertaken. The site was visited to provide infection prevention and control (IPC) support. Isolates were emm typed. A total of 38 residents and 29 staff in contact with resident cases were provided with antibiotic chemoprophylaxis. Seven additional staff with no direct resident contact also received chemoprophylaxis after finding one probable localised GAS infection among them. No more iGAS cases subsequently occurred.Site visit recommendations included advice on terminal cleaning and cleaning of shared equipment, as well as strengthening staff education on hand hygiene and masking. All isolates were of emm subtype 18.12, a subtype not previously detected in Ireland. Key outbreak control measures were rapid delivery of IPC support and chemoprophylaxis. Emm18 is infrequently associated with GAS infections.

Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

Streptococcal Pharyngitis: Rapid Evidence Review.

Group A beta-hemolytic streptococcal pharyngitis is a common infection responsible for more than 6 million office visits in the United States annually. Only 10% of adults seeking care for a sore throat have group A beta-hemolytic streptococcal pharyngitis; however, 60% or more are prescribed antibiotics. Guidelines recommend using clinical decision rules to assess the risk of group A beta-hemolytic streptococcal infection, followed by rapid antigen testing if a diagnosis is unclear, before prescribing antibiotics. Fever, tonsillar exudate, cervical lymphadenitis, and patient ages of 3 to 15 years increase clinical suspicion. A cough is more suggestive of a viral etiology. The limited history used in these decision rules is amenable to virtual visits. After a negative rapid antigen test result, a throat culture is recommended in children and adolescents. Penicillin and amoxicillin are first-line antibiotics, with a recommended course of 10 days; first-generation cephalosporins are recommended for patients with nonanaphylactic allergies to penicillin. There is significant resistance to azithromycin and clarithromycin in some parts of the United States. Steroids are not recommended for symptomatic treatment. Patients with worsening symptoms after appropriate antibiotic initiation or with symptoms lasting 5 days after the start of treatment should be reevaluated. Tonsillectomy is rarely recommended as a preventive measure: seven episodes of streptococcal pharyngitis in 1 year, five episodes in each of the past 2 years, or three episodes in each of the past 3 years are commonly used thresholds for considering surgery.

The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies.

Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3's Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.

Group A Streptococcus pyogenes in wastewater: Applicability of wastewater-based epidemiology for monitoring the prevalence of GAS pharyngitis during the late COVID-19 pandemic phase.

Streptococcus pyogenes, Group A Streptococcus (GAS), is a human pathogen that causes a spectrum of diseases from mild to severe, including GAS pharyngitis, a common acute respiratory disease in developed countries. Although wastewater-based epidemiology (WBE) has been extensively used to monitor viral pathogens such as severe acute respiratory syndrome coronavirus 2, its applicability to S. pyogenes remains unexplored. This study was conducted to investigate the feasibility of detecting and quantifying S. pyogenes in wastewater by quantitative polymerase chain reaction (qPCR) and evaluate the applicability of WBE for monitoring the prevalence of GAS pharyngitis. A total of 52 grab influent samples were collected from a wastewater treatment plant in Japan once a week between March 2023 and February 2024. The samples were centrifuged, followed by nucleic acid extraction and qPCR for the S. pyogenes-specific genes speB and spy1258. Of the 52 samples, 90 % and 81 % were positive for speB and spy1258 genes, respectively, indicating the feasibility of S. pyogenes for wastewater surveillance. However, the percentage of quantifiable samples for speB gene was significantly higher in winter than in spring and summer. Similarly, the concentrations of both genes in wastewater samples were significantly higher in winter (speB, 4.1 ± 0.27 log10 copies/L; spy1258, 4.1 ± 0.28 log10 copies/L; One-way ANOVA, p < 0.01) than in spring and summer. Higher concentrations and detection ratios of S. pyogenes genes were observed during increased GAS pharyngitis cases in the catchment. Significant moderate correlations were observed between target gene concentrations and reported GAS pharyngitis cases. This study enhances the understanding role of WBE in monitoring and managing infectious diseases within communities.

Why Does the E1219V Mutation Expand T-Rich PAM Recognition in Cas9 from Streptococcus pyogenes?

Popular RNA-guided DNA endonuclease Cas9 from Streptococcus pyogenes (SpCas9) recognizes the canonical 5'-NGG-3' protospacer adjacent motif (PAM) and triggers double-stranded DNA cleavage activity. Mutations in SpCas9 were demonstrated to expand the PAM readability and hold promise for therapeutic and genome editing applications. However, the energetics of the PAM recognition and its relation to the atomic structure remain unknown. Using the X-ray structure (precatalytic SpCas9:sgRNA:dsDNA) as a template, we calculated the change in the PAM binding affinity in response to SpCas9 mutations using computer simulations. The E1219V mutation in SpCas9 fine-tunes the water accessibility in the PAM binding pocket and promotes new interactions in the SpCas9:noncanonical T-rich PAM, thus weakening the PAM stringency. The nucleotide-specific interaction of two arginine residues (i.e., R1333 and R1335 of SpCas9) ensured stringent 5'-NGG-3' PAM recognition. R1335A substitution (SpCas9R1335A) completely disrupts the direct interaction between SpCas9 and PAM sequences (canonical or noncanonical), accounting for the loss of editing activity. Interestingly, the double mutant (SpCas9R1335A,E1219V) boosts DNA binding affinity by favoring protein:PAM electrostatic contact in a desolvated pocket. The underlying thermodynamics explain the varied DNA cleavage activity of SpCas9 variants. A direct link between the energetics, structures, and activity is highlighted, which can aid in the rational design of improved SpCas9-based genome editing tools.